Specific alien plant species predominantly deliver nectar sugar and pollen but are not preferentially visited by wild pollinating insects in suburban riparian ecosystems

Abstract The invasion of alien plants has been rapidly proceeding worldwide due to urbanisation. This might be beneficial to wild pollinating insects, since some alien plant species have large flowers and/or long flowering periods, which can increase nectar sugar and pollen availability. To determine the relative contribution of alien plants to floral resource supply and whether resource‐rich alien plants, if any, serve as an important food source of pollinating insects, we performed year‐round field observations in suburban riverbanks. We quantified the per‐unit‐area availability of nectar sugar and pollen delivered by alien and native flowering species and counted wild flower visitors (bees and wasps, hoverflies and butterflies) per plant species. The available nectar sugar and pollen per area were predominantly delivered by a few specific alien species, and the relative contribution of other species to floral resource provision was low throughout the period that wild flower visitors were observed. Nonetheless, the resource‐rich alien plants were not visited by as many insects as expected based on their contribution to resource provision. Rather, on a yearly basis, these plants received equal or even fewer visits than other flowering species, including resource‐poor natives. We show that despite their great contribution to the gross floral resource supply, resource‐rich alien plants do not serve as a principal food source for wild pollinating insects, and other plants, especially natives, are still needed to satisfy insect demand. For the conservation of pollinating insects in suburban ecosystems, maintaining floral resource diversity would be more beneficial than having an increase in gross floral resources by allowing the dominance of specific alien plants.


Basic strategy
Floral resources have generally been quantified in terms of sugar mass and pollen volume (Comba et al. 1999;Torres 2000;Corbet et al. 2001;Baude et al. 2016;Hicks et al. 2016;Nottebrock et al. 2017;Nakamura and Kudo 2019). Sugar mass can be determined based on the volume of nectar and the sugar concentration of the nectar (Corbet et al. 2001;Baude et al. 2016;Hicks et al. 2016), while pollen volume can be determined based on the size and total number of pollen grains (Hicks et al. 2016;Nakamura and Kudo 2019). Accordingly, we first empirically measured the following four variables: the volume of nectar (μl), the sugar concentration (g sucrose/100 g nectar), the number of pollen grains and the major and minor axes of a pollen grain (μm), after which we calculated sugar mass (μg) and pollen volume (μl) based on the measured variables.
The empirical measurement of the variables described above was carried out at the level of a single flower for all except Asteraceae and Trifolium of which field observations of floral abundance was conducted at the flower head level (Table A). Following Baude et al. (2016), for species belonging to Asteraceae producing capitula (flower heads), we first measured the resources in a floret and then counted the number of florets per head, and subsequently the species mean amount of resources per head was calculated as the product of the mean number of florets per head and the mean amount of resources in a floret. As an exception, for an Asteraceae species Youngia japonica, measurement was conducted direcely at the level of the flower head including all florets in a head (Table S1). For species belonging to Trifolim (Fabaceae) that produce densely globose inflorescences (also called flower heads), we counted the number of flowers per inflorescence and measured the resources in a flower. We then quantified the species mean resources per inflorescence following the same procedure applied for Asteraceae. Exceptions to this strategy were made in T. campestre and T. dubium, in which the measurement of pollen was conducted at the inflorescence level, as in the case of Y. japonica (Table S1).  flower sample was used in one measurement event, but in some cases, multiple samples were used to obtain a measurable amount of nectar, and the amount was divided by the number of flower samples used. In each measurement event, we used as many tubes as necessary to empty the sample(s). Sampling was conducted between 6:00 and 16:30 on rainless days. Prior to sampling, the flowers were bagged with a fine net to avoid insect visits and were allowed to accumulate nectar for 2 h. As written in the main manuscript, this duration of nectar accumulation was shorter than that in previous studies (i.e., 24 h in Baude et al. 2016;Hicks et al. 2016;Tew et al. 2021) and would not be appropriate for assessing absolute nectar productivity of species; however, because our focus was on capturing the relative differences among species in contribution to available floral resources, we considered that this accumulation duration can be justified for this study.
We determined the nectar volume by measuring the length of the microcapillary tube occupied by the extracted nectar. The sugar concentration of the sampled nectar was then immediately measured using a hand-held sucrose refractometer modified for small sample volumes (Bellingham and Stanley Eclipse 45-81 and 45-82, Tunbridge Wells, UK; Figure A).
For some species producing small flowers/florets, we applied the procedure described by Baude et al. (2016) and Hicks et al. (2016); first, we rinsed a flower or floret with 2 μl of distilled water applied at the location of the nectaries using a micropipette and then measured the sugar concentration of the resulting solution after waiting for 1 min (for the list of species, see Table S1). Because sugar concentration measurements are temperature dependent, we calibrated the value read with the refractometer to remove the effects of temperature according to the manual provided by the manufacturer (https://www.scientificlabs.co.uk/handlers/libraryFiles.ashx?filename=Manuals_R_REF1050_ C.pdf, last accessed on 30 May 2021). We adopted the calibrated sugar concentration value to calculate sugar mass.
Following previous studies ( For each species, we calculated the mean lengths of the major and minor axes of the pollen grains and the mean number of pollen grains per flower/floret/head. The species mean volume of a pollen grain (μm 3 ) was calculated as V = 4/3πAB 2 , where A is half of the mean length of the major axis of the pollen grain, and B is half of the mean length of the minor axis (Hicks et al. 2006). The species mean pollen volume (μl) per flower or floret (or per flower head for Y. japonica, T. campestre and T. dubium) was then obtained by multiplying the mean

Data verification
The validity of the species mean values of nectar sugar mass and pollen volume calculated in this study was assessed by comparing our data with the data from previously published studies. We performed standardised major axis linear regression (sma function in the "smart" library) using the statistical package R ver. 3.6.3 (R Core Team 2020). For the data on the sugar mass per flower or floret in units of µg, we compared the species mean values of 13 shared species across six families between our study and the study by Baude et al. (2016) (for the list of species used for validation, see Table S1). There was a significant positive correlation between the two datasets with a slope that was reasonably close to 1 (slope = 0.63, intercept = 7.86, R 2 = 0.93, P < 0.001; Figure C (a)). As shown by the slope of 0.63, our data were slightly smaller than that in Baude et al. (2016); this was reasonable because the duration of nectar accumulation we applied was shorter than Baude et al. (2016). This result indicates that our sugar mass values were consistent with the published values. For the pollen volume per flower or floret in units of µl, we compared our species mean values with those of Hicks et al. (2016). Because of the limited number of shared species (eight species across five families; for the list of species used for validation, see Table S1), the correlation was not significant (slope = 1.67, intercept = 0.03, R 2 = 0.46, P = 0.066), and the uncertainty was large ( Figure C (b)). However, some species showed similar pollen volumes in the two studies; for example, 0.005 µl and 0.007 µl for Sisymbrium officinale, 0.184 µl and 0.146 µl for Lotus corniculatus, and 0.007 µl and 0.003 µl for Persicaria maculosa in our study and that of Hicks et al. (2016), respectively. These results support potential concordance between the two datasets.